Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14211—Erythrovirus, e.g. B19 virus
  • C12N2750/14222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates to modified Parvovirus B19 capsid proteins and particles, their uses and the preparation of me ⁇ dicaments including vaccines for the treatment of parvoviurs B19 associated diseases.
• Parvovirus B19 was discovered in 1975 as cause of systemic in ⁇ fections of adults who either were asymptomatic or had mild non specific symptoms such as headache, pyrexia, malaise, fatigue, and myalgia (1, 2, 3) .
• parvovirus B19 was identified as the etiological agent of erythema infectiosum, also known as fifth disease (4) .
• the virus is spread worldwide but restricted exclusively to human hosts. With respect to regional differences about 40 to 60 percent of the population displays antibodies against viral proteins at an age of 20 years. The infection is endemic, however reaches regional outbreaks during late winter and spring time. The virus is very stable. In general it is transmitted orally from individuals with acute infection (5) .
• the virus may be transmit ⁇ ted through parenterally administered blood and blood derived products and vertically from mother to fetus (6, 7, 8, 9) .
• Mas ⁇ sive productive replication of parvovirus B19 takes place in erythroid progenitor cells (10) . Therefore during acute infec ⁇ tion the virus load is extremely high and up to 10 13 particles and/or virus genomes may be present per milliliter of the pe ⁇ ripheral blood.
• the virus binds to erythrocyte precursor cells using the blood group antigen P (globoside) as the cellular re ⁇ ceptor for adsorption (11) .
• parvovirus B19 infection is without clinical symptoms or may result in a flu like disease. Particularly children may suffer from erythema infectiosum (fifth disease) , a self limit- ing rash illness (18, 19, 20) . Parvovirus B19 has been further associated with a wide spectrum of diseases (Table 1) : Transient anemia, leucocytopenia or thrombocytopenia may occur without re ⁇ quiring any therapy. However, in some patients severe thrombocy ⁇ topenia, pure red cell aplasia or pancytopenia were observed.
• parvovirus B19 infection may induce a broad spectrum of autoimmune phenomena.
• Autoimmune cy- topenias are well known hematological disorders that may affect any bone-marrow cell lineage. In the mass majority of cases only one of the cell lines is affected.
• autoim ⁇ mune-mediated destruction of neutrophil granulocytes and throm ⁇ bocytes due to persistent parvovirus B19 infection is known (31) .
• Parvovirus B19 has been identified as a possible trigger in some cases of immune thrombocytopenia (32, 33, 34, 35) .
• the clinical spectrum of autoimmune joint disease ranges from mild arthralgias to severe necrotizing vasculitis (Table 1) .
• arthralgias and arthritis have been commonly reported. B19-infection may produce a symmet ⁇ rical peripheral polyarthropathy in adults. Symptoms are usually self-limited but may persist for several months. Occasionally viremia combined with IgM/IgG-antibodies against structural pro ⁇ teins VPl and/or VP2 has been shown in patients with long last ⁇ ing polyarthralgia/polyarthritis . Some of them met the criteria of rheumatoid arthritis (RA) (26, 37) . However development of RA after acute parvovirus infection seems to be rare (38, 39) .
• RA rheumatoid arthritis
• Parvovirus B19 infection has been associated with various forms of vasculitis, collagenosis and may mimick systemic lupus ery ⁇ thematosus (SLE) in children and adults (39, 45, 46) . Similar to the situation in arthritis patients also in SLE-patients parvo ⁇ virus B19 infection has been described both as the agent causing or triggering the rheumatic disease (47, 48, 49, 50, 51) . Re ⁇ cently, an association between persistent parvovirus B19 infec ⁇ tion and the production of anti-phospholipid antibodies in pedi ⁇ atric and adult patients with rheumatic disease was described (52) .
• the antiphospholipid syndrome (APS) (53) is, like some parvovirus B19 infections, characterized by a wide variety of hemocytopenic and vaso-occlusive manifestations. Additionally recurrent fetal loss and the association with autoantibodies di ⁇ rected against negatively charged phospholipids and protein co- factors, mainly ⁇ 2-glycoprotein-I are important features of the APS.
• the hypothesis regarding a common pathogenicity of the anti-phospholipid syndrome and the autoimmunity features ob ⁇ served in B19-infection is supported not only by the close asso ⁇ ciation between the presence of anti-phospholipid antibodies and parvovirus B19 infection but also by the similarity in the pre ⁇ senting of clinical symptoms in patients with parvovirus B19 in ⁇ fection and patients with the APS.
• the icosahedral capsid of parvovirus B19 consist of two struc ⁇ tural proteins, VPl (83kDa) and VP2 (58 kDa) , which are identi ⁇ cal except for 227 amino acids (aa) at the amino-terminal end of the VPl protein (the VPl unique region) .
• a phospholipase A2 mo ⁇ tif is present in the amino acid sequence of the VPl unique re ⁇ gion spanning positions 130 to 195 (Phospholipase A2 active cen ⁇ ter) (54) .
• One of the pathogenic mechanisms involved in trigger ⁇ ing the production of anti-phospholipid antibodies might be the phospholipase-A2-like activity observed in the VPl-unique region of the structural protein VPl of parvovirus B19 (54) .
• This en ⁇ zyme activity is present in infectious B19-particles, in recom ⁇ binant empty capsids consisting of VP1/VP2-proteins and in preparations of purified VPl-uinique region.
• the problem underlining the present inventions is to provide me ⁇ dicaments such as a parvovirus B19 vaccine for the prevention and/or treatment of parvovirus B19 associated diseases with minimal side effects.
• a modified VPl-capsid protein of parvovirus B19 having a reduced phospholipase A2-like enzyme activity as compared to the wild type VPl-capsid protein of parvovirus B19 having the amino acid sequence of Seq.ID No 1.
• the natural phospholipase activity is reduced (which includes complete abolition of the activity) . Therefore, regions of the protein which are involved in reduction of this phospholipase activity can be altered ac ⁇ cording to the present invention.
• These changes compared to wild type sequence may be introduced by amino acid exchanges, dele ⁇ tions or insertions. Reduction of phospholipase activity can easily be assessed by the skilled man in the art e.g. by relying on the methodology disclosed herein, especially according to the test described in Fig. 2 of Dorsch et al, 2002 (54) .
• the changes should not significantly alter the immu ⁇ nological performance of the vaccine (i.e.
• the amino acid residues in the VPl-unique region of the VPl- capsid protein are altered. This region is highly conserved in all three geno ⁇ types of parvovirus B19 that have been identified until today
• Genotype 1 100%
• Genotype 2 70%
• genotype 3 59%.
• Reference to wild type activity means that the closest genotype is selected for comparison of wild type mutant
• whole VPl protein may be subject to the introduction of mutations for reducing or eliminating phospholipase A2-like activity of this protein.
• mutations outside the VPl unique region amino acids 100-220, specifically 130 to 195 have usually a low, if any, reduction potential on the phospho ⁇ lipase activity
• mutations inside this VPl unique region are preferred according to the present invention.
• mutations according to the present invention may be introduced, e.g. by amino trun ⁇ cation or by mutations in the region of amino acid 50 - 90, es ⁇ pecially 59 - 81.
• a mutation fulfills the expected re ⁇ duction of phospholipase activity can easily be addressed by the combination of the expression of the mutant with a functional activity assay.
• Such phospholipase activity tests are well available in the art and also described herein.
• Specifically preferred sites of mutation are around the Ca 2+ binding amino acids (Tyr(130) , Gly(132) , Gly(134) , Asp (154)) or around the catalytic network (His(153) , Tyr(157) , Tyr(168)) , in ⁇ cluding the phospholipid binding (Lys(162)) , i.e. amino acids 125-140 and 150-175, especially 127-137, 152-160 and 165 to 171 (see also SeqID No. 1 and Fig. 2 for sequences) .
• Preferred exchanges are the exchanges usually used in enzyme ac ⁇ tivity reducing mutation strategies, e.g. replacing an amino acid with a functional side chain (His, Lys, Tyr, Asp, GIu, Ser, Thr, Asn, GIn, etc.) with an amino acid with a non functional side chain of the same or different size (Ala, Phe, Leu, Pro, lie, etc.) or with a different functional side chain (e.g. Asn ⁇ Asp, GIn + Ser, etc.) . Sometimes, also a slight difference is sufficient (VaI - ⁇ Ala, Ser - ⁇ Thr, etc.) .
• the altered amino acid residues reside in the phospholi- pase A2 center of the VPl-unique region of the VPl-capsid pro ⁇ tein.
• one or more of the conserved amino acid residues in the active centre of the phospholipase A2 are altered.
• Amino acid residues at position 153: histidine,- 157: tyrosine; 162: lysine; 168: tyrosine,- 195: aspartic acid have been de ⁇ scribed as parts of the catalytic triad of bovine pancreatic phospholipase A2 and to be involved in substrate orientation and specificity (58, 59) .
• one or more of the amino acid residues are altered at position 153: histidine; position 157: tyrosine; position 162: lysine; and/or position 168: tyrosine.
• histidine 153 is altered into alanine
• - tyrosine 157 is altered into phenylala ⁇ nine
• lysine 162 is altered into leucine
• tyrosine 168 is altered into phenylalanine.
• the mutation according to the present invention should be as minimal as possible (in order not to alter immuno ⁇ genic properties too much) : usually a low number of amino acid substitutions (6, 5, 4, 3, 2 or, preferably, 1) and a short in ⁇ sertion or deletion (30, 20, 10, 5 or 1 amino acids) should be sufficient to efficiently reduce or remove phospholipase activ ⁇ ity.
• a low number of amino acid substitutions (6, 5, 4, 3, 2 or, preferably, 1)
• a short in ⁇ sertion or deletion (30, 20, 10, 5 or 1 amino acids) should be sufficient to efficiently reduce or remove phospholipase activ ⁇ ity.
• longer insertions, deletion (50, 100, 200 amino ac ⁇ ids) or a higher number of substitutions are not excluded, as long as the immunogenic properties are not significantly changed.
• multiple mutations are also less preferred due to cost aspects.
• the mutants have similar immunogenic properties or not can easily be determined e.g. by reacting with specific antibod ⁇ ies or by determing in vitro or in vivo ability to induce immune responses.
• the mutants should exhibit (with or with ⁇ out specific adjuvants) about 50% of in vivo immunogenicity as the wild type (without adjuvants, or preferably, with the same adjuvant as the mutant) , determined by at least one scientifi ⁇ cally applied and (by peer review) accepted method, e.g. anti ⁇ body binding, ELISpot assays, etc. (see also: example section of the present application) .
• the modified VPl-unique re ⁇ gion or the modified VPl-capsid protein of the present invention results in a reduced phospholipase A2-like enzyme activity as compared to the wild type VPl-capsid protein.
• the reduction is at least 30% compared to the wild type (i.e. 70% or less of wild type activity) .
• Reference activity of wild type and mutant are preferably determined according to a standardized phospholi ⁇ pase A2-like enzyme activity test, especially according to fig. 2 of (54) .
• mutants which completely lack this activity are most suitable for vaccine use and safety according to the present invention.
• the alternation of the amino acid residues is made by site-directed mutagenesis.
• an isolated nucleic acid molecule encoding the modified VPl-capsid protein provided by the present invention is provided.
• a recombinant expression vector comprising the nucleic acid molecules encoding the modified VPl-unique region and the modified VPl-capsid protein provided by the present invention.
• the recombinant expression vector according to the present invention further comprises VP2-capsid protein of parvo ⁇ virus B19 especially wherein the protein is being expressable together with the modified VP-I capsid.
• VP2-capsid protein of parvo ⁇ virus B19 especially wherein the protein is being expressable together with the modified VP-I capsid.
• the com ⁇ bined expression of the VPl and VP2 proteins using two vectors that are introduced into the cells has its advantages for spe ⁇ cific purposes.
• the recombinant expression vector according to the present invention comprises a fusion product in that the modified VPl-unique region or the modified VPl-capsid protein is fused to the VP2-capsid protein.
• a host cell comprising the recombinant expression vec ⁇ tor according to the present invention.
• the host cell is E.coli, yeast, or an animal cell.
• the host cell is Saccharomyces cerevisiae. This species of yeast has been used for two decades to produce recom ⁇ binant HBsAg-particles that protect against hepatitis B virus infection. The use of recombinant S.cerevisiae derived HBV vac ⁇ cines has been shown to be safe, side effects are observed only very rarely.
• a process of producing the modified VPl-unique region or the modified VPl-capsid protein or the fusion product of the modified VPl-capsid protein and VP2-capsid protein by transform ⁇ ing a host cell, expressing the VPl-capsid (and optionally the VP2-capsid) , recovering the protein(s) , optionally as virus-like particles, using the host cell according to the present inven ⁇ tion.
• the modified VPl-unique region and/or the modified VPl-capsid protein and/or the fusion product of the modified VPl-capsid protein and VP2-capsid protein are isolated and/or purified.
• the modified VPl-unique region, and/or the modified VPl-capsid protein or the fusion product of the modified VPl- unique region and VP2- capsid protein obtainable by the process according to the present invention.
• the modified VPl-unique region and/or the modified VPl-protein or the fusion product of the modified VPl-unique re ⁇ gion and/or the modified VPl-protein and VP2-capsid protein for use as medicaments.
• the modified VPl-unique region, the modified VPl-protein with or without VP2-capsid protein for the manufac ⁇ ture of a medicament for the treatment against parvovirus B19 infection and/or parvovirus B19 associated autoimmune and rheu ⁇ matic diseases.
• the treatment is against Arthralgias; Arthritis,- more preferably Monoarthritis, Oligoarthritis, Polyarthritis, Rheuma- toid arthritis and/or Juvenile idiopathic arthritis; Systemic lupus erythematosus (SLE) ; Vasculitis, more preferably Leuko- clastic vasculitis, Purpura Henlein-Schoenoch, Papular-purpuric gloves-and-socks syndrome (PPGSS) , Kawasaki disease, Giant cell arteritis (GCA) , Polyarteritis nodosa and/or Wegener's granulo ⁇ matosis; Dermatomyositis; Autoimmune neutropenia; Autoimmune thrombocytpenia,- Idiopathic thrombocytopenic purpura (ITP) ; Autoimmune hemolytic anemia,- and/or Virus-associated hemophago- cy
• the modified VPl-unique region, the modified VPl capsid protein or the fusion product of the modified VPl- unique region and VP2-capsid protein according to the present invention in an assay for detecting antibodies directed against the B19 virus protein VPl in a sample to be tested.
• the host cells of the present invention in an assay for detecting antibodies directed against the B19 virus protein VPl in a sample to be tested.
• the recombinant virus-like particles consisting of the modified VPl-capsid protein according to the present invention with or without VP2-capsid protein.
• the recombinant virus-like particles consisting of the fusion product of the modified VPl-unique region, the modi ⁇ fied VPl-capsid protein and VP2-capsid protein according to the present invention.
• those re ⁇ combinant virus-like particles can be used for the manufacture of a medicament for the treatment against parvovirus B19 infec ⁇ tion and/or parvovirus B19 associated autoimmune and rheumatic diseases.
• the treatment is preferably against Arthralgias; Arthritis,- more preferably Monoarthritis, Oligoarthritis, Polyarthritis, Rheuma ⁇ toid arthritis and/or Juvenile idiopathic arthritis; Systemic lupus erythematosus (SLE) ; Vasculitis, more preferably Leuko- clastic vasculitis, Purpura Henlein-Schoenoch, Papular-purpuric gloves-and-socks syndrome (PPGSS) , Kawasaki disease, Giant cell arteritis (GCA) , Polyarteritis nodosa and/or Wegener's granulo ⁇ matosis; Dermatomyositis; Autoimmune neutropenia; Autoimmune thrombocytpenia,- Idiopathic thrombocytopenic purpura (ITP) ; Autoimmune hemolytic anemia,- and/or Virus-associated hemophago- cy
• a pharmaceutical composition especially a vaccine preparation for inducing an immune response which provides pro ⁇ tection against the human parvovirus B19, comprising the modi ⁇ fied VPl-unique region, the modified VPl-capsid protein accord ⁇ ing to the present invention, with or without the VP2-capsid protein.
• a pharmaceutical composition especially a vaccine preparation for inducing an immune response which provides pro ⁇ tection against the human parvovirus B19, comprising the fusion product of the modified VPl-capsid protein according to the pre ⁇ sent invention.
• a pharmaceutical composition especially a vaccine preparation for inducing an immune response which pro ⁇ vides protection against the human parvovirus B19, comprising the recombinant virus-like particles according to the present invention.
• the pharmaceutical compositions according to the present invention are further comprising one or more carriers and/or adjuvants suitable for vaccination purposes.
• compositions provided by the present invention may further comprise an im- munostimulatory substance, preferably selected from the group comprising a polycationic compound, preferably a polycationic polymer, more preferably a polycationic peptide, especially polyarginine, polylysine or an antimicrobial peptide.polymers,- immunostimulatory deoxynucleotides (ODNs) ; peptides containing at least two LysLeuLys motifs,- neuroactive compounds, especially human growth hormone,- alumn, Freund' s complete or incomplete ad ⁇ juvants or combinations thereof.
• ODNs immunostimulatory deoxynucleotides
• the present vaccine preferably comprises an modified VPl capsid protein according to the present in ⁇ vention as antigen a peptide comprising a sequence R I -XZXZ N XZX-R 2 , whereby N is a whole number between 3 and 7, preferably 5, X is a positively charged natural and/or non-natural amino acid residue, Z is an amino acid residue selected from the group consisting of L, V, I, F and/or W, and Ri and R 2 are se ⁇ lected independantly one from the other from the group consist ⁇ ing of -H, -NH 2 , -COCH 3 , -COH, a peptide with up to 20 amino acid residues or a peptide reactive group or a peptide linker with or without a peptide; X-R 2 may be an amide, ester or thioester of the C-terminal amino acid residue of the peptide (in the follow ⁇ ing also referred to as "Peptide A”) and/or
• ODN immunostimulatory oligodeoxynucleic acid molecule
• Rl is selected from hypoxanthine and uracile, any X is 0 or S, any NMP is a 2 ' deoxynucleoside monophosphate or monothiophos- phate, selected from the group consisting of deoxyadenosine- , deoxyguanosine- , deoxyinosine- , deoxycytosine- , deoxyuridine- , deoxythymidine- , 2-methyl-deoxyinosine- , 5-methyl-deoxycytosine- , deoxypseudouridine- , deoxyribosepurine- , 2-amino- deoxyribosepurine- , 6-S-deoxyguanine- , 2-dimethyl- deoxyguanosine- or N-isopentenyl-deoxyadenosine-monophosphate or -monothiophosphate,
• NUC is a 2 ' deoxynucleoside, selected from the group consisting of deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycyto ⁇ sine-, deoxyinosine-, deoxythymidine-, 2-methyl-deoxyuridine- , 5-methyl-deoxycytosine- , deoxypseudouridine- , deoxyribosepurine- , 2-amino-deoxyribosepurine- , 6-S-deoxyguanine-, 2-dimethyl- deoxyguanosine- or N-isopentenyl-deoxyadenosine, a and b are integers from 0 to 100 with the proviso that a + b is between 4 and 150, and
• B and E are common groups for 5 ' or 3 ' ends of nucleic acid molecules (in the following also referred to as "I-/U-ODN") .
• the present vaccine may further contain other sub ⁇ stances, e.g. suitable pharmaceutically acceptable diluents or carrier, buffer or stabilising substances, etc..
• the vaccine according to the present invention may also contain other or additional adjuvants, especially an Al(OH) 3 adjuvant (Alum) .
• Alum as meant herein includes all forms of Al 3+ based adjuvants used in human and animal medicine and research. Especially, it includes all forms of aluminum hydroxide as defined in R ⁇ mpp, 10th Ed. pages 139/140, gel forms thereof, aluminum phosphate, etc..
• the polycationic peptides or compound to be used according to the present invention may be any polycationic compound which shows the characteristic effect according to the WO 97/30721.
• Preferred polycationic compounds are selected from basic poly ⁇ peptides, organic polycations, basic polyaminoacids or mixtures thereof . These polyaminoacids should have a chain length of at least 4 amino acid residues.
• WO 97/30721 e.g. polyethyleneimine
• WO 99/38528 e.g. polyethyleneimine
• these polypeptides contain between 20 and 500 amino acid residues, especially between 30 and 200 residues.
• polycationic compounds may be produced chemically or re- combinantly or may be derived from natural sources.
• Cationic (poly)peptides may also be polycationic anti-bacterial microbial peptides. These (poly)peptides may be of prokaryotic or eukaryotic origin or may be produced chemically or recombi- nantly. Peptides may also belong to the class naturally occur ⁇ ring antimicrobial peptides. Such host defense peptides or de ⁇ fensives are also a preferred form of the polycationic polymer according to the present invention. Generally, a compound allow ⁇ ing as an end product activation (or down-regulation) of the adaptive immune system, preferably mediated by APCs (including dendritic cells) is used as polycationic polymer. Furthermore, also neuroactive compounds, such as (human) growth hormone (as described e.g. in WO01/24822) may be used as immu- nostimulants (Immunizers) .
• Polycationic compounds derived from natural sources include HIV- REV or HIV-TAT (derived cationic peptides, antennapedia pep ⁇ tides, chitosan or other derivatives of chitin) or other pep ⁇ tides derived from these peptides or proteins by biochemical or recombinant production.
• Other preferred polycationic compounds are cathelin or related or derived substances from cathelicidin, especially mouse, bovine or especially human cathelicidins and/or cathelicidins.
• Related or derived cathelicidin substances contain the whole or parts of the cathelicidin sequence with at least 15-20 amino acid residues. Derivations may include the substitution or modification of the natural amino acids by amino acids which are not among the 20 standard amino acids.
• catheli ⁇ cidin molecules may be introduced into such catheli ⁇ cidin molecules.
• These cathelicidin molecules are preferred to be combined with the antigen/vaccine composition according to the present invention.
• these cathelin molecules sur ⁇ prisingly have turned out to be also effective as an adjuvant for a antigen without the addition of further adjuvants. It is therefore possible to use such cathelicidin molecules as effi ⁇ cient adjuvants in vaccine formulations with or without further immunactivating substances.
• the vaccine according to the present invention preferably con ⁇ tains as Peptide A KLKL 5 KLK and/or as I-/U-ODN oligo d(IC)i 3 (The combination of Peptide A and Oligo-d(IC) 13 is also referred as IC31) . These two substances are specifically advantageous ac ⁇ cording to the present invention.
• the vaccine according to the present invention may contain an oligodeoxynucleotide containing a CpG-motif as immunomodulating nucleic acids.
• the immunomodulating nucleic acids to be used ac ⁇ cording to the present invention can be of synthetic, prokary- otic and eukaryotic origin. In the case of eukaryotic origin, DNA should be derived from, based on the phylogenetic tree, less developed species (e.g. insects, but also others) .
• the immunogenic oligodeoxynu- cleotide (ODN) is a synthetically produced DNA-molecule or mix ⁇ tures of such molecules.
• ODNs such as thiophosphate substituted analogues (thiophosphate residues substitute for phosphate) as for example described in US patents US 5,723,335 and US 5,663,153, and other derivatives and modifi ⁇ cations, which preferably stabilize the immunostimulatory compo ⁇ sition(s) but do not change their immunological properties, are also included.
• a preferred sequence motif is a six base DNA mo ⁇ tif containing an (unmethylated) CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines (5 ' -Pur-Pur-C-G-Pyr-Pyr-3 ' ) .
• the CpG motifs contained in the ODNs according to the present invention are more common in microbial than higher vertebrate DNA and display differences in the pattern of methylation. Sur ⁇ prisingly, sequences stimulating mouse APCs are not very effi ⁇ cient for human cells. Preferred palindromic or non-palindromic ODNs to be used according to the present invention are disclosed e.g.
• ODNs/DNAs may be produced chemically or recombinantly or may be derived from natural sources. Preferred natural sources are insects.
• the vaccine according to the present invention may preferably contain a polycationic peptide and an oligodeoxynucleotide con ⁇ taining a CpG-motif in combination.
• the combination of CpG-ODN and polycationic peptide has improvement effects in vaccine com ⁇ positions, which are comparable to the effects of the combina ⁇ tion of Peptide A and 1-/U-ODNs and cannot only be combined with Peptide A and 1-/U-ODNs but even be used instead of them.
• cathelicidin-derived antimicrobial peptides or derivatives thereof have an immune response stimulating activity and there ⁇ fore constitute highly effective type 1 inducing adjuvants (Im- munizers) .
• Main sources of antimicrobial peptides are granules of neutrophils and epithelial cells lining the respiratory, gas- tro-intestinal and genitourinary tracts. In general they are found at those anatomical sites most exposed to microbial inva ⁇ sion, are secreted into internal body fluids or stored in cyto ⁇ plasmic granules of professional phagocytes (neutrophils) .
• a type 1 inducing adjuvant that is able to strongly enhance the immune response to a specific co-administered antigen and therefore constitutes a highly ef ⁇ fective adjuvant
• Peptide A comprising a sequence R 1 -XZXZ N XZX-R 2 .
• a specifically preferred peptide is KLKLLLLLKLK. Besides naturally occuring antimicrobial peptides, synthetic an ⁇ timicrobial peptides have been produced and investigated.
• the synthetic antimicrobial peptide KLKLLLLLKLK-NH2 was shown to have significant chemotherapeutic activity in Staphylococcus aureus-infected mice,- human neutrophils were activated to pro ⁇ cute the superoxide anion (02 " ) via cell surface calreticulin. The exact number and position of K and L was found to be criti ⁇ cal for the antimicrobial activity of the synthetic peptide.
• the present invention is especially beneficial if the vaccine is administered subcutaneousIy, intramusculary, intradermally or transdermally.
• other application forms such as paren ⁇ teral, intravenously, intranasally, oral or topical application, are also suitable for the present invention.
• the Parvovirus antigen according to the present invention may be mixed with an adjuvant (Immunizer) (composition) or otherwise specifically formulated e.g. as liposome, retard formulation, etc..
• adjuvant immunosorbent
• composition or otherwise specifically formulated e.g. as liposome, retard formulation, etc.
• the vaccines according to the present invention may be adminis ⁇ tered to an individual in effective amounts known to the skilled man in the art. Optimisation of antigen amount and Immunizer amount can be started from established amounts and using avail ⁇ able methods .
• a diagnostic kit comprising a modified VPl-unique re ⁇ gion, a modified VPl-capsid protein according to the present in ⁇ vention and ancillary reagents.
• a diagnostic kit comprising a fusion product of the modified VPl-capsid protein according to the present in ⁇ vention and ancillary reagents.
• a diagnostic kit comprising a recombinant virus-like particles according to the present invention and ancillary rea ⁇ gents .
• the modified VPl-unique region, the modified VPl-capsid protein according to the present invention with or without VP2-capsid protein as an agent to modify the activity of host phospholipase A2 activity, e.g. by gene therapy on using antisense KNA or RNAi.
• the use of the recombinant virus-like particles accord ⁇ ing to the present invention as an agent to modify the activity of host phospholipase A2 activity is provided.
• Fig. 1 shows an immunological comparison of wt and mutant pro ⁇ tein
• Fig. 2 shows an alignment of Parvovirus VPl protein.
• VP1/VP2 antigens without enzymatic activity can be achieved by alteration of the residues that are part of the active centre by site-directed mutagenesis.
• site-directed mutagenesis Despite the fact that the overall size of cellular enzymes with Ca 2+ -dependent phospholipase A2 activity and the viral VPl-unique region are different, alignments comparing the amino acid sequences re ⁇ vealed a number of conserved residues in the region that repre ⁇ sents the active centre of the enzyme. conserveed amino acids were observed at the following positions:
• Residue 153 histidine,- residue 157: tyrosine; residue 162: lysine; residue 168: tyrosine; residue 195: aspartic acid.
• the respective amino acids have been described as parts of the cata ⁇ lytic triad of bovine pancreatic phospholipase A2 and to be in ⁇ volved in substrate orientation and specificity. Therefore the alteration of these residues in the VPl-unique region by site- directed mutagenesis was performed using polymerase chain reac ⁇ tion with mutated primer and overlap extension as initially de ⁇ scribed by Ho and coworkers (56) .
• GCA Giant cell arteritis
• VAHS Autoimmune hemolytic anemia Virus-associated hemophagocytic syndrome
• Table 2 Enzmyatic phopholipase A2-like activity in the VPl- unique region of parvovirus B19 and variants constructed by site-directed mutagenesis.
• mice Inoculation of mice.
• Groups of 5 female Balc/C mice were inocu ⁇ lated with 50 ug of purified preparations of the VPl-unique re ⁇ gion/wildtype and the VPl-unigue region/H153A in emulsion with complete' s Freund's Adjuvans.
• Retrobulbar blood samples were taken at days 0, 3, 7, 10, 14, 18 and 28 after inoculation. The sera were tested for the presence of IgG antibodies against the VPl-unique region/wildtype in ELISA assays.
• the sequences encoding the VPl-unique re- gion/wildtype and the VPl-unique region/H153A were cloned into the T7-expression vector pET21a_int in fusion with an intein and a chitin-binding domain as described previously (Dorsch et al . , 2001, Dorsch et al . , 2002) .
• the constructs were introduced into the E. coli strain BL21. Bacteria were inoculated with LB (luria broth) medium containing lOO ⁇ g/ml ampicillin and incubated at 37 0 C.
• the column was washed with 2 volumes of 2OmM HEPES, ImM EDTA, 10OmM NaCl, pH8,5, 8 volumes of 2OmM HEPES, ImM EDTA, 2mM NaCl, pH8,5 and 2 volumes of 2OmM HEPES, ImM EDTA, 10OmM NaCl, pH8,5. Afterwards the protein was eluted using 3 volumes of 5OmM DTT in buffer 2OmM HEPES, ImM EDTA, 10OmM NaCl, pH8,5. Fractions were tested for the recombinant proteins by SDS-PAGE and silver staining.
• ELISA-assay Microtiter plates (Maxisorb, Nunc, Wiesbaden, FRG) were coated over night with purified protein ⁇ VPl-unique re- gion/wildtype, 100 ng/well) in 0.2 M NaCO 3 buffer, pH 9.2 con ⁇ taining 0.15 M NaCl. Sera were used in dilutions of 1:100 in PBS/0.5% Tween-20 and IgG-antibodies were detected using HRP- coupled polyclonal rabbit anti-mouse IgG as second antibodies (ditution 1:5000 in PBS/0.5% Tween-20, Dako, Hamburg FRG) and TMB as substrate, the optical density was determined at 450 nm.
• Antibodies in ⁇ quiz against the variant VPl-unique region/H153A have the ca ⁇ pacity to bind to the VPl-unique region/ ' wildtype which had used as antigen in the ELISA indicating an high degree of cross reac ⁇ tivity. Mice that were inoculated with PBS in emulsion with com ⁇ plete Freund' s adjuvans did not develop any significant amounts of VPl-specific antibodies.
• the wildtype VPl-unique region antigen and the mutant antigen (Hisl53Ala) were inoculated in mice.
• VPl-specific antibody pro ⁇ duction was analysed by ELISA. No differences were devised in the capacity of both antigens to elicit VPl-specific antibody production.
• Antibodies against the mutant antigen Hisl53Ala were similarly active to bind to the wildtype VPl-unique region and vice versa. This indicates that the mutant Hisl53Ala variant of the VPl-unique region of parvovirus B19 has a comparable capac ⁇ ity to elicit the production of antibodies as the wildtype pro ⁇ tein domain.
• main neutralising epitopes are known to be located in protein parts different from the active centre of the viral phospholipase A2-like enzyme and were not affects by any of the introduced mutations effects on the protein's immuno- genicity are unlikely (57) .
• Figure 1 The development of IgG-antibodies against the VPl- unique region/wildtype. Groups of 5 mice were inoculated with either the VPl-unigue region/wildtype, the VPl-unigue re- gion/H153A or PBS. Serum samples taken at days after inoculation as indicated were tested in ELISA using the VPl-unique region as antigen. Average values obtained from testing the individual samples of 5 mice were determined in each case.

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